A Preliminary Study Of The Transcription Factor KLF5 Promoting Differential Exosomal MiRNA Expression In Bladder Carcinogenesis

PurposeThe occurrence and development of bladder cancer(BCa)is a complex process regulated by multiple genes.The transcription factor KLF5 is a member of the KLFs family.It has been shown that the expression of KLF5 can be regulated by miRNAs and KLF5 can also regulate the expression of miRNAs,both of which are jointly involved in the progression of malignant tumors.In this study,we analyzed the effects of KLF5 on exosomal miRNA expression and secretion in bladder cancer cells based on exosome micro RNA microarray and q RT-PCR.The screening and identification of exosomal miRNAs that can be used for early diagnosis,prognostic analysis and targeted therapy of bladder cancer are expected to improve the clinical efficacy of bladder cancer.MethodOur group’s previous study showed that the level of KLF5 transcription factor was significantly increased in the serum of bladder cancer patients,which was positively correlated with tumor progression.And by analyzing the total RNA of each group of cells after transfection of BIU-87 bladder cancer cells with KLF5 adenovirus overexpression vector,interference vector and control vector,five miRNAs with significant differences were screened out,and after validation at the cellular level and tissue level,it was found that miR-145-5p,miR-214-5p,miR-375 and miR-148-3p expression levels were down-regulated,and miR-9-5p expression levels were up-regulated.Based on this study,we further investigated the effect of high KLF5 expression on exosome expression in bladder cancer cells.1.exosome extraction and identification The KLF5 adenovirus overexpression vector(pAd-KLF5),interference vector(si-KLF5)and control vector(pAd)were constructed and transfected with bladder cancer BIU-87 cells for 48 h.The expression level of KLF5 protein in transfected cells was measured using protein blotting(western blot)to assess the transfection efficiency.The exosomes were extracted from pAd-KLF5 and pAd group cells after transfection by ultracentrifugation,and the particle size and concentration of exosomes were analyzed by nanoparticle tracking analysis(NTA)and transmission electron microscope(TEM).The morphology of joint fluid exosomes was observed by Nanoparticle tracking analysis(NTA),transmission electron microscope(TEM)and western blot method was used to detect specific immunoproteins on the surface of exosomes for the identification of extracted exosomes.2.Validation of exosomal miRNA analysis Extract the total RNA from exosomes of pAd-KLF5 and pAd-transfected bladder cancer BIU-87 cells,and select samples with total RNA mass >10μg,concentration >200ng/μl,RIN≥8,28S/18S≥1.5 for micro RNA library construction.Micro RNA microarrays were prepared and the prepared micro RNA microarrays were analyzed for standard micro RNA microarray data.Human U6 was used as the internal reference gene,and relative quantification was performed by using RNA extraction,reverse transcription process and fluorescence quantitative PCR assay,which in turn verified the exosomal miRNAs differentially expressed in each group of cells.3.Clinical case validation Thirty cases of bladder cancer diagnosed by histopathology in the Department of Urology,Huaihe Hospital of Henan University and treated by total cystectomy were selected,and bladder cancer tissues and corresponding paracancerous tissues were obtained intraoperatively,immediately placed in liquid nitrogen and stored in a refrigerator at-80 ℃.RNA was extracted from bladder cancer and paraneoplastic tissues by Trizol reagent,and the expression levels of differentially expressed miRNAs in bladder cancer and paraneoplastic tissues were verified by q RT-PCR.Serum exosomes of both groups were extracted by ultracentrifugation and exosomal RNA was obtained,and the expression levels of differentially expressed miRNAs were verified by q RT-PCR in the serum exosomes of bladder cancer patients and controls.ResultsIn this study,five miRNAs with significant differences in bladder cancer cell exosomes were screened: miR-145-5p,miR-214-5p,miR-375,miR-9-5p,and miR-148-3p.By validation,it was found that compared with BIU-87 cells transfected with the control vector,the expression levels of miR-145-5p,miR-214-5p,miR-375,and miR-148-3p were down-regulated in the exosomes of BIU-87 cells transfected with the KLF5 overexpression vector,while the expression levels of miR-9-5p were up-regulated.145-5p,miR-214-5p,miR-375 and miR-148-3p expression levels were down-regulated,while the expression level of miR-9-5p was up-regulated.The results of bladder cancer tissue validation suggested that miR-145-5p,miR-214-5p and miR-375 expression was significantly downregulated in bladder cancer tissues compared to normal tissues adjacent to the cancer.Similarly in bladder cancer patient sera and control serum exosomes,miR-145-5p,miR-214-5p and miR-375 expression was significantly downregulated in serum exosomes.ConclusionIn this study,we screened and identified differential miRNAs closely associated with high KLF5 expression in bladder cancer cell exosomes by exosomal micro RNA microarray and and q RT-PCR techniques,and verified by clinical bladder cancer tissues and serum exosome levels,and found that miR-145-5p,miR-214-5p and miR-375 were significantly down-regulated in bladder cancer tissues and bladder cancer patients The expression of miR-145-5p,miR-214-5p and miR-375 was significantly down-regulated in bladder cancer tissues and serum exosomes.The exosomal miRNAs identified in this study can be used for the early diagnosis and prognosis of bladder cancer,and also provide a possible experimental basis for the application of KLF5 in the clinical targeting therapy of bladder cancer.